Mixed chimerism and tolerance

ABSTRACT

A method of inducing tolerance without whole body irradiation.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.09/374,498, filed on Aug. 13, 1999, now U.S. Pat. No. 6,412,492, whichis a division of U.S. application Ser. No. 08/855,705, filed on May 8,1997, now U.S. Pat. No. 6,006,752, which is a continuation-in-part ofU.S. Provisional Application No. 60/017,099, filed May 9, 1996, all ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The invention relates to tissue and organ transplantation.

SUMMARY OF THE INVENTION

The invention provides methods of inducing tolerance to foreignantigens. The methods feature preparative regimens which minimize oreliminate the need for hematopoietic space-creating irradiation,especially, preparative whole body irradiation. In particular, it hasbeen discovered that the administration of a relatively large number ofstem cells, combined with the creation of thymic space, can allow theinduction of tolerance without the need for whole body irradiation(WBI).

Accordingly, the invention features a method of inducing tolerance in arecipient mammal of a first species to a graft from a donor mammal of asecond species. The method includes: introducing, e.g., by intravenousinjection, into the recipient mammal, hematopoietic stem cells; andpreferably, implanting the graft in the recipient. The hematopoieticcells are believed to prepare the recipient for the graft that follows,by inducing tolerance at both the B-cell and T-cell levels.

The recipient mammal can be, by way of example, a human. The donormammal can be, by way of example, a swine, e.g., a miniature swine. Thegraft is preferably from a discordant species. The graft preferablyexpresses a major histocompatibility complex (MHC) antigen, preferably aclass II antigen. In particularly preferred embodiments the recipient isa primate, e.g., a human, and the donor is a swine, e.g., a miniatureswine.

As is discussed elsewhere herein, the inventors have discovered thatthis method can be practiced without the administration of hematopoieticspace-creating irradiation, e.g., whole body irradiation. Whole bodyirradiation is often used in the art to create hematopoietic space andthus promote engraftment, chimerism, and tolerance. The need forhematopoietic space-creating irradiation can be reduced or entirelyeliminated by the inclusion of one or more of the following steps in themethod:

(1) Administering a sufficiently large number of donor hematopoieticcells to the recipient such that, donor stem cells engraft, give rise tomixed chimerism, and induce tolerance, preferably the stem cells areadministered either in combination with one or more of the treatmentsdisclosed herein, e.g., (2), (3), or (4) described immediately below;

(2) Administering hematopoietic space creating antibodies or drugs tothe recipient. E.g., administering an inhibitor of cell proliferation,e.g., DSG, or an anti-metabolite, e.g. brequinar, or an anti-T cellantibody, e.g., one or both of an anti-CD4 or anti-CD8 antibody.

(3) providing treatments (other than whole body irradiation) whichpromote engraftment and the formation of mixed chimerism by enhancingthe ability of donor cells to compete with host bone marrow cells, e.g.,administering stromal cells or administering donor specific growthfactors or cytokines, e.g., where the donor is a miniature swine,administering one or more of swine SCF, swine IL-3, or swine GM-SCF, tothe recipient.

(4) creating thymic space in the recipient, e.g., by irradiating thethymus of the recipient, e.g., by administering between 100 and 1,000,more preferably between 300 and 700, e.g., 700 rads, of thymicirradiation, or by administering anti-T cell antibodies in sufficientdose to inactivate thymocytes. Other methods for the creation of thymicspace include: the administration of steroids, corticosteroids,brequinar, or an immune suppressant chemical or drug, e.g., rapamycin,cyclosporin, or FK506. Treatment to create thymic space should beadministered, or at least begun, prior to the administration ofhematopoietic stem cells. An effective treatment should deplete singlepositive thymocytes to an extent that engraftment and the formation ofmixed chimerism is optimized in the absence of the creation ofhematopoietic space, e.g., hematopoietic space created by whole bodyirradiation. In preferred embodiments the subject's single positivethymocytes are depleted by at least 20, 40, 60, or 80%. Treatments whichresult in between 10 and 90% depletion are preferred. The length of thetreatment will vary with dosage and the effectiveness of the agent butwill generally be less than 60, 30, or 15 days. The treatment shouldlast at least 7, and more preferably 10, or 14 days in length. Inpreferred courses of treatment, e.g., the administration of animmunosupressive chemical or drug, e.g., cyclosporine, should lastbetween 7 and 30 days. The treatment, e.g., the administration ofcyclosporin, should be started at a time such that it is completed priorto the administration of stem cells. Administration of the agent shouldbe on a daily basis or as needed to maintain a level of the agent whichallows the desired level of depletion. A particularly preferredtreatment is the administration of an immunosuppresive chemical, e.g.,cyclosporin, for more than 7 and less than 30 days. A useful regimen inrodents is 20 mg/kg/day cyclosporin for 14 days ending on the third daybefore administration of stem cells.

Thus, in preferred embodiments a quantity of hematopoietic stem cellssufficient to induce tolerance, without the need for hematopoieticspace-creating irradiation, is administered to the recipient. Inpreferred embodiments the number of donor hematopoietic cells is atleast twice, is at least equal to, or is at least 75, 50, or 25% asgreat as, the number of bone marrow cells found in an adult of therecipient species. In preferred embodiments the number of donorhematopoietic stem cells is at least twice, is at least equal to, or isat least 75, 50, or 25% as great as, the number of bone marrowhematopoietic stem cells found in an adult of the recipient species. Inthe case where an inbred population of the donor species exists, e.g.,where the donor species is miniature swine, the number of availabledonor cells is not limited to the number of cells which can be obtainedfrom a single animal. Thus, in such cases, the donor cells administeredto the recipient can come from more than one, e.g., from two, three,four, or more animals. As is discussed below the donor stem cells can beprovided in two or more separate administrations.

In preferred embodiments, mixed chimerism is induced in the recipientand the state of mixed chimerism is formed in the absence of theinduction of hematopoietic space, e.g., in the absence of hematopoieticspace created by space creating irradiation, e.g., whole bodyirradiation.

The number of donor cells administered to the recipient can be increasedby either increasing the number of stem cells provided in a particularadministration or by providing repeated administrations of donor stemcells.

Repeated stem cell administration can promote engraftment, mixedchimerism, and long-term deletional tolerance in graft recipients. Thus,the invention also includes methods in which multiple hematopoietic stemcell administrations are provided to a recipient. Multipleadministration can substantially reduce or eliminate the need forhematopoietic space-creating irradiation. Administrations can be givenprior to, at the time of, or after graft implantation. In preferredembodiments multiple administrations of stem cells are provided prior tothe implantation of a graft. Two, three, four, five, or moreadministrations can be provided. The period between administrations ofhematopoietic stem cells can be varied. In preferred embodiments asubsequent administration of hematopoietic stem cell is provided: atleast two days, one week, one month, or six months after the previousadministration of stem cells, when the recipient begins to show signs ofhost lymphocyte response to donor antigen; when the level of chimerismdecreases; when the level of chimerism falls below a predeterminedvalue; when the level of chimerism reaches or falls below a level wherestaining with a monoclonal antibody specific for a donor PBMC antigen isequal to or falls below staining with an isotype control which does notbind to PBMC's, e.g. when the donor specific monoclonal stains less than1-2% of the cells; or generally, as is needed to maintain a level ofmixed chimerism sufficient to maintain tolerance to donor antigen.

One or more post graft-implantation-administrations of donor stem cellscan also be provided to minimize or eliminate the need for irradiation.Post graft administration of hematopoietic stem cell can provided: atleast two days, one week, one month, or six months after the previousadministration of stem cells; at least two days, one week, one month,six months, or at any time in the life span of the recipient after theimplantation of the graft; when the recipient begins to show signs ofrejection, e.g., as evidenced by a decline in function of the graftedorgan, by a change in the host donor specific antibody response, or by achange in the host lymphocyte response to donor antigen; when the levelof chimerism decreases; when the level of chimerism falls below apredetermined value; when the level of chimerism reaches or falls belowa level where staining with a monoclonal antibody specific for a donorPBMC antigen is equal to or falls below staining with an isotype controlwhich does not bind to PBMC's, e.g. when the donor specific monoclonalstains less than 1-2% of the cells; or generally, as is needed tomaintain tolerance or otherwise prolong the acceptance of a graft.

When multiple stem cell administrations are given one or more of theadministrations can include a number of donor hematopoietic cells whichis at least twice, is equal to, or is at least 75, 50, or 25% as greatas, the number of bone marrow cells found in an adult of the recipientspecies; include a number of donor hematopoietic stem cells which is atleast twice, is equal to, or is at least 75, 50, or 25% as great as, thenumber of bone marrow hematopoietic stem cells found in an adult of therecipient species.

In preferred embodiments the method includes inactivating natural killercells, preferably graft reactive or xenoreactive, e.g., swine reactive,NK cells, of the recipient mammal. This can be accomplished, e.g., byintroducing into the recipient mammal an antibody capable of binding tonatural killer cells of the recipient mammal. The administration ofantibodies, or other treatment to inactivate natural killer cells, canbe given prior to introducing the hematopoietic stem cells into therecipient mammal or prior to implanting the graft in the recipient. Thisantibody can be the same or different from an antibody used toinactivate T cells.

In preferred embodiments the method includes inactivating T cells,preferably graft reactive or xenoreactive, e.g., swine reactive, T cellsof the recipient mammal. This can be accomplished, e.g., by introducinginto the recipient mammal an antibody capable of binding to T cells ofthe recipient mammal. The administration of antibodies, or othertreatment to inactivate T cells, can be given prior to introducing thehematopoietic stem cells into the recipient mammal or prior toimplanting the graft in the recipient. This antibody can be the same ordifferent from an antibody used to inactivate natural killer cells.

One source of anti-NK antibody is anti-human thymocyte polyclonalanti-serum. Preferably, a second anti-mature T cell antibody can beadministered as well, which lyses T cells as well as NK cells. Lysing Tcells is advantageous for both bone marrow and graft survival. Anti-Tcell antibodies are present, along with anti-NK antibodies, inanti-thymocyte anti-serum. Repeated doses of antibodies, e.g., anti-NKor anti-T cell antibodies, may be preferable. Monoclonal preparationscan be used in the methods of the invention.

In preferred embodiments the recipient does not receive treatments whichstimulate the release of a cytokine by mature T cells. E.g., therecipient should not receive a substance, e.g., a steroid drug, e.g.,Prednisone (17, 21-dihydroxypregna-1, 4-diene-3, 11, 20-trione), at adosage or concentration which stimulates the release of a cytokine bymature T cells in the recipient. Preferably, the recipient is free ofsuch treatment from the time stem cells are first administered until thegraft is implanted or until mixed chimerism and tolerance isestablished.

In preferred embodiments the method includes the administration of ashort course of help reducing treatment, e.g., a drug or other chemicalagent, which induces tolerance to unmatched class I and/or minorantigens on the graft which is introduced into the recipient. The shortcourse of help reducing treatment, e.g., a short course of high dosecyclosporine, is generally administered at the time at the graft isintroduced into the recipient. The duration of the short course of helpreducing treatment is approximately equal to or is less than the periodrequired for mature T cells of the recipient species to initiaterejection of an antigen after first being stimulated by the antigen; inmore preferred embodiments, the duration is approximately equal to or isless than two, three, four, five, or ten times, the period required fora mature T cell of the recipient species to initiate rejection of anantigen after first being stimulated by the antigen. These methods aredescribed in more detail in co-owned application Ser. No. 08/458,720,filed Jun. 1, 1995, which is hereby incorporated by references. Methodsof Ser. No. 08/458,720 can be combined with the methods describedherein.

Other preferred embodiments include: the step of introducing into therecipient mammal, donor species-specific stromal tissue, preferablyhematopoietic stromal tissue, e.g., fetal liver or thymus. In preferredembodiments: the stromal tissue is introduced simultaneously with, orprior to, the hematopoietic stem cells; the hematopoietic stem cells areintroduced simultaneously with, or prior to, the antibody.

Other preferred embodiments include treatments to further inactivaterecipient T cells, particularly thymic or lymph node thymocytes or Tcells. Thymic or lymph node thymocytes or T cells might otherwiseinhibit the engraftment or survival of the administered cells. Suchinactivation can be accomplished by one or more of: irradiating thethymus of the recipient mammal with a dose of radiation sufficient toinactivate thymocytes, e.g., 100-1,000, more preferably between 300 and700, e.g., about 350 or 700 rads of thymic irradiation; administeringone or repeated doses of an anti-T cell or anti-thymocyte antibody; oradministering to the recipient a short course of an immunosuppressantchemical or drug, as is described herein. Inactivation of thymocytes orT cells can be performed prior to hematopoietic stem cell or grafttransplantation. In preferred embodiments the method includesdiminishing or inhibiting thymocyte or T cell activity, preferably theactivity of thymic or lymph node T cells by administering to therecipient a short course of an immunosuppressive agent, e.g., a chemicalor drug, e.g., cyclosporine, sufficient to inactivate thymocytes or Tcells, preferably thymic or lymph node T cells. The duration of theshort course of immunosuppressive agent is: approximately equal to 30days; approximately equal to or less than 8-12 days, preferably about 10days; approximately equal to or less than two, three, four, five, or tentimes the 8-12 or 10 day period. The short course can begin: before orat about the time the treatment to induce tolerance is begun, e.g., atabout the time stem cells are introduced into the recipient; on the daythe treatment to induce tolerance is begun, e.g., on the day stem cellsare introduced into the recipient; within 1, 2, 4, 6, 8, 10, or 30 daysbefore or after the treatment to induce tolerance is begun, e.g., within1, 2, 4, 6, 8, 10, or 30 days before or after stem cells are introducedinto the recipient. The short course of an immunosuppressive can beadministered in conjunction with an anti-T cell antibody The shortcourse of an immunosuppressive should be sufficient in concentration andduration to inactivate T cells, e.g., thymic or lymph node T cells,which would not be inactivated by antibody-based inactivation of Tcells, e.g., inactivation by intravenous administrations of ATGantibody, or similar, preparations.

Other embodiments include (optionally): the step of, prior tohematopoietic stem cell transplantation, creating hematopoietic space,e.g., by irradiating the recipient mammal with low dose, e.g., less than400, preferably less than 300, more preferably less than 200 or 100rads, whole body irradiation to deplete or partially deplete the bonemarrow of the recipient. As is discussed herein this treatment can bereduced or entirely eliminated.

Other preferred embodiments include: the step of, preferably prior tohematopoietic stem cell transplantation, depleting natural antibodiesfrom the blood of the recipient mammal. Depletion can be achieved, byway of example, by contacting the recipients blood with an epitope whichabsorbs preformed anti-donor antibody. The epitope can be coupled to aninsoluble substrate and provided, e.g., as an affinity column. E.g., anα1-3 galactose linkage epitope-affinity matrix, e.g., matrix boundlinear B type VI carbohydrate, can be used to deplete naturalantibodies. Depletion can also be achieved by hemoperfusing an organ,e.g., a liver or a kidney, obtained from a mammal of the donor species.(In organ hemoperfusion antibodies in the blood bind to antigens on thecell surfaces of the organ and are thus removed from the blood.)

Other preferred embodiments include those in which: the same mammal ofthe second species is the donor of one or both the graft and thehematopoietic cells; and the antibody is an anti-human thymocytepolyclonal anti-serum, obtained, e.g., from a horse or pig.

In preferred embodiments, the method includes the step of introducinginto the recipient a graft obtained from the donor which is obtainedfrom a different organ than the hematopoietic stem cells, e.g., a heart,pancreas, liver, or kidney.

Methods of the invention which substantially reduce or eliminate theneed for hematopoietic space creating irradiation can be used whenimplanting allogeneic stem cells. Accordingly, in another aspect, theinvention features a method of inducing tolerance in a recipient mammalof a first species to a graft from a donor mammal of the same species.The recipient mammal can be, by way of example, a primate, e.g., ahuman. The graft preferably expresses a major histocompatibility complex(MHC) antigen, preferably a class II antigen.

The method includes: introducing, e.g., by intravenous injection, intothe recipient mammal, hematopoietic stem cells; and preferably,implanting the graft in the recipient. The hematopoietic cells arebelieved to prepare the recipient for the graft that follows, byinducing tolerance at both the B-cell and T-cell levels.

This method can be practiced without the administration of hematopoieticspace-creating irradiation, e.g., whole body irradiation. Whole bodyirradiation is often used in the art to create hematopoietic space andthus promote engraftment, chimerism, and tolerance. The need forhematopoietic space-creating irradiation can be reduced or entirelyeliminated by inclusion of one or more of the following steps in themethod:

(1) Administering a sufficiently large number of donor hematopoieticcells to the recipient such that donor stem cells engraft, give rise tomixed chimerism, and induce tolerance, preferably, the stem cells areadministered in combination with one or more of the treatments disclosedherein, e.g., (2) or (3) immediately below;

(2) Administering hematopoietic space creating antibodies or drugs tothe recipient. E.g., administering an inhibitor of cell proliferation,e.g., DSG, or an anti-metabolite, e.g. brequinar, or an anti-T cellantibody, e.g., one or both of an anti-CD4 or anti-CD8 antibody.

(3) creating thymic space in the recipient, e.g., by irradiating thethymus of the recipient, e.g., by administering between 100 and 1,000,more preferably between 300 and 700, e.g., 700 rads, of thymicirradiation, or by administering anti-T cell antibodies in sufficientdose to inactivate thymocytes. Other methods for the creation of thymicspace include: the administration of steroids, corticosteroids,brequinar or an immune suppressant chemical or drug, e.g, rapamycin,cyclosporin, or FK506. Treatment to create thymic space should beadministered, or at least begun, prior to the administration ofhematopoietic stem cells. An effective treatment should deplete singlepositive thymocytes to an extent that engraftment and the formation ofmixed chimerism is optimized in the absence of the creation ofhematopoietic space, e.g., hematopoietic space created by whole bodyirradiation. In preferred embodiments the subject's single positivethymocytes are depleted by at least 20, 40, 60, or 80%. Treatments whichresult in between 10 and 90% depletion are preferred. The length of thetreatment will vary with dosage and the effectiveness of the agent butwill generally be less than 60, 30, or 15 days. The treatment shouldlast at least 7, and more preferably 10, or 14 days in length. Inpreferred courses of treatment, e.g., the administration of animmunosupressive chemical or drug, e.g., cyclosporine, should lastbetween 7 and 30 days. The treatment, e.g., the administration ofcyclosporin, should be started at a time such that it is completed priorto the administration of stem cells. Administration of the agent shouldbe on a daily basis or as needed to maintain a level of the agent whichallows the desired level of depletion. A particularly preferredtreatment is the administration of an immunosuppresive chemical, e.g.,cyclosporin, for more than 7 and less than 30 days. A useful regimen inrodents is 20 mg/kg/day cyclosporin for 14 days ending on the third daybefore administration of stem cells.

Thus, in preferred embodiments a quantity of hematopoietic stem cellssufficient to induce tolerance, without the need for hematopoieticspace-creating irradiation, is administered to the recipient. Inpreferred embodiments the number of donor hematopoietic cells is atleast twice, is at least equal to, or is at least 75, 50, or 25% asgreat as, the number of bone marrow cells found in an adult of therecipient species. In preferred embodiments the number of donorhematopoietic stem cells is at least twice, is at least equal to, or isat least 75, 50, or 25% as great as, the number of bone marrowhematopoietic stem cells found in an adult of the recipient species.

In preferred embodiments, mixed chimerism is induced in the recipientand the state of mixed chimerism is formed in the absence of theinduction of hematopoietic space, e.g., in the absence of hematopoieticspace created by space creating irradiation, e.g., whole bodyirradiation.

The number of donor cells administered to the recipient can be increasedby either or both of increasing the number of stem cells provided in aparticular administration or by providing repeated administrations ofdonor stem cells.

Repeated stem cell administration can promote engraftment, mixedchimerism, and long-term deletional tolerance in graft recipients. Thus,the invention also includes methods in which multiple hematopoietic stemcells administrations are provided to a recipient. Multipleadministration can substantially reduce or eliminate the need forhematopoietic space-creating irradiation. Administrations can be givenprior to, at the time of, or after graft implantation. In preferredembodiments multiple administrations of stem cells are provided prior tothe implantation of a graft. Two, three, four, five, or moreadministrations can be provided. The period between administrations ofhematopoietic stem cells can be varied. In preferred embodiments asubsequent administration of hematopoietic stem cell is provided: atleast two days, one week, one month, or six months after the previousadministration of stem cells; when the recipient begins to show signs ofhost lymphocyte response to donor antigen; when the level of chimerismdecreases; when the level of chimerism falls below a predeterminedvalue; when the level of chimerism reaches or falls below a level wherestaining with a monoclonal antibody specific for a donor PBMC antigen isequal to or falls below staining with an isotype control which does notbind to PBMC's, e.g. when the donor specific monoclonal stains less than1-2% of the cells; or generally, as is needed to maintain a level ofmixed chimerism sufficient to maintain tolerance to donor antigen.

One or more post graft-implantation-administrations of donor stem cellscan also be provided to minimize or eliminate the need for irradiation.Post graft administration of hematopoietic stem cell can provided: atleast two days, one week, one month, or six months after the previousadministration of stem cells; at least two days, one week, one month,six months, or at any time in the life span of the recipient after theimplantation of the graft; when the recipient begins to show signs ofrejection, e.g., as evidenced by a decline in function of the graftedorgan, by a change in the host donor specific antibody response, or by achange in the host lymphocyte response to donor antigen; when the levelof chimerism decreases; when the level of chimerism falls below apredetermined value; when the level of chimerism reaches or falls belowa level where staining with a monoclonal antibody specific for a donorPBMC antigen is equal to or falls below staining with an isotype controlwhich does not bind to PBMC's, e.g. when the donor specific monoclonalstains less than 1-2% of the cells; or generally, as is needed tomaintain tolerance or otherwise prolong the acceptance of a graft.

When multiple stem cell administrations are given one or more of theadministrations can: include a number of donor hematopoietic cells whichis at least twice, is equal to, or is at least 75, 50, or 25% as greatas, the number of bone marrow cells found in an adult of the recipientspecies; include a number of donor hematopoietic stem cells which is atleast twice, is equal to, or is at least 75, 50, or 25% as great as, thenumber of bone marrow hematopoietic stem cells found in an adult of therecipient species.

In preferred embodiments the method includes inactivating natural killercells, preferably graft reactive or donor reactive NK cells, of therecipient mammal. This can be accomplished, e.g., by introducing intothe recipient mammal an antibody capable of binding to natural killercells of the recipient mammal. The administration of antibodies, orother treatment to inactivate natural killer cells, can be given priorto introducing the hematopoietic stem cells into the recipient mammal orprior to implanting the graft in the recipient. This antibody can be thesame or different from an antibody used to inactivate T cells.

In preferred embodiments the method includes inactivating T cells,preferably graft reactive or donor reactive T cells, of the recipientmammal. This can be accomplished, e.g., by introducing into therecipient mammal an antibody capable of binding to T cells of therecipient mammal. The administration of antibodies, or other treatmentto inactivate T cells, can be given prior to introducing thehematopoietic stem cells into the recipient mammal or prior toimplanting the graft in the recipient. This antibody can be the same ordifferent from an antibody used to inactivate natural killer cells

One source of anti-NK antibody is anti-human thymocyte polyclonalanti-serum. Preferably, a second anti-mature T cell antibody can beadministered as well, which lyses T cells as well as NK cells. Lysing Tcells is advantageous for both bone marrow and graft survival. Anti-Tcell antibodies are present, along with anti-NK antibodies, inanti-thymocyte anti-serum. Repeated doses of antibodies, e.g., anti-NKor anti-T cell antibodies, may be preferable. Monoclonal preparationscan be used in the methods of the invention.

In preferred embodiments the recipient does not receive treatments whichstimulate the release of a cytokine by mature T cells. E.g., therecipient should not receive a substance, e.g., a steroid drug, e.g.,Prednisone (17,21-dihydroxypregna-1,4-diene-3,11,20-trione), at a dosageor concentration which stimulates the release of a cytokine by mature Tcells in the recipient. Preferably, the recipient is free of suchtreatment from the time stem cells are first administered until thegraft is implanted or until mixed chimerism and tolerance isestablished.

In preferred embodiments the method includes the administration of ashort course of help reducing treatment, e.g., a drug or other chemical,which induces tolerance to unmatched class I and/or minor antigens onthe graft which is introduced into the recipient. The short course ofhelp reducing treatment, e.g., a short course of high dose cyclosporine,is generally administered at the time at the graft is introduced intothe recipient. The duration of the short course of help reducingtreatment is approximately equal to or is less than the period requiredfor mature T cells of the recipient species to initiate rejection of anantigen after first being stimulated by the antigen; in more preferredembodiments, the duration is approximately equal to or is less than two,three, four, five, or ten times, the period required for a mature T cellof the recipient species to initiate rejection of an antigen after firstbeing stimulated by the antigen. These methods are described in moredetail in co-owned application Ser. No. 08/458,720, filed Jun. 1, 1995,which is hereby incorporated by references. Methods of Ser. No.08/458,720 can be combined with the methods described herein.

Other preferred embodiments include treatments to further inactivaterecipient T cells, particularly thymic or lymph node thymocytes or Tcells. Thymic or lymph node thymocytes or T cells might otherwiseinhibit the engraftment or survival of the administered cells. Suchinactivation can be accomplished by one or more of: irradiating thethymus of the recipient mammal with a dose of radiation sufficient toinactivate thymocytes, e.g., 100-1,000, more preferably between 300 and700, e.g., about 350 or 700 rads of thymic irradiation; administeringone or repeated doses of an anti-T cell or anti-thymocyte antibody; oradministering to the recipient a short course of an immunosuppressantchemical or drug, as is described herein. Inactivation of thymocytes orT cells can be performed prior to hematopoietic stem cell or grafttransplantation. In preferred embodiments the method includesdiminishing or inhibiting thymocyte or T cell activity, preferably theactivity of thymic or lymph node T cells by administering to therecipient a short course of an immunosuppressive agent, e.g.,cyclosporine, sufficient to inactivate thymocytes or T cells, preferablythymic or lymph node T cells. The duration of the short course ofimmunosuppressive agent is: approximately equal to 30 days;approximately equal to or less than 8-12 days, preferably about 10 days;approximately equal to or less than two, three, four, five, or ten timesthe 8-12 or 10 day period. The short course can begin: before or atabout the time the treatment to induce tolerance is begun, e.g., atabout the time stem cells are introduced into the recipient; on the daythe treatment to induce tolerance is begun, e.g., on the day stem cellsare introduced into the recipient; within 1, 2, 4, 6, 8, 10, or 30 daysbefore or after the treatment to induce tolerance is begun, e.g., within1, 2, 4, 6, 8, 10, or 30 days before or after stem cells are introducedinto the recipient. The short course of an immunosuppressive can beadministered in conjunction with an anti-T cell antibody. The shortcourse of an immunosuppressive should be sufficient in concentration andduration to inactivate T cells, e.g., thymic or lymph node T cells,which would not be inactivated by antibody-based inactivation of Tcells, e.g., inactivation by intravenous administrations of ATGantibody, or similar, preparations.

Other embodiments include (optionally): the step of, prior tohematopoietic stem cell transplantation, creating hematopoietic space,e.g., by irradiating the recipient mammal with low dose, e.g., less than400, preferably less than 300, more preferably less than 200 or 100rads, whole body irradiation to deplete or partially deplete the bonemarrow of the recipient. As is discussed herein this treatment can bereduced or entirely eliminated.

In another aspect, the invention features a method of inducing toleranceto, or prolonging acceptance of, a graft from a donor mammal. The methodincludes: diminishing or inhibiting thymocyte or T cell activity,preferably the activity of thymic or lymph node T cells, byadministering to the recipient, a short course of an immunosuppressiveagent, e.g., a drug or other chemical, e.g., cyclosporine, sufficient toinactivate thymocytes or T cells, preferably thymic or lymph node Tcells. The duration of the short course of immunosuppressive agent is:approximately equal to 30 days; approximately equal to or less than 8-12days, preferably about 10 days; approximately equal to or less than two,three, four, five, or ten times the 8-12 or 10 day period. The shortcourse can begin: before the introduction of door tissue into therecipient, preferably and will end 1, 2, 4, 6, 8, 10, or 30 days beforeintroduction of donor tissue.

In preferred embodiments: the recipient is a primate, e.g., a human, andthe graft is an allograft; the recipient is a primate, e.g., a human,and the donor is from a second species, e.g., a second primate speciesor a swine.

This method can be combined with any of the other methods describedherein.

“Discordant species combination”, as used herein, refers to two speciesin which hyperacute rejection occurs when a graft is grafted from one tothe other. Generally, discordant species are from different orders,while non-discordant species are from the same order. For example, ratsand mice are non-discordant concordant species. Concordant speciescombinations do not exhibit hyperacute rejection.

“Graft”, as used herein, refers to a body part, organ, tissue, or cells.Organs such as liver, kidney, heart or lung, or other body parts, suchas bone or skeletal matrix, tissue, such as skin, intestines, endocrineglands, or progenitor stem cells of various types, are all examples ofgrafts.

“Help reducing agent”, as used herein, is an agent, e.g., animmunosuppressive drug, which results in the reduction of cytokinerelease. Examples of help reducing agents are cyclosporine, FK-506, andrapamycin. Anti-T cell antibodies, because they can eliminate T cells,are not preferred for use as help reducing agents. A help reducing agentmust be administered in sufficient dose to give the level of inhibitionof cytokine release which will result in tolerance. The help reducingagent should be administered in the absence of treatments which promotecytokine, e.g., IL-2, release. Putative help reducing agents can beprescreened by in vitro or in vivo tests, e.g., by contacting theputative agent with T cells and determining the ability of the treated Tcells to release a cytokine, e.g., IL-2. The inhibition of cytokinerelease is indicative of the putative agent's efficacy as a helpreducing agent. Such prescreened putative agents can then be furthertested in a kidney transplant assay. In a kidney transplant assay aputative help reducing agent is tested for efficacy by administering theputative agent to a recipient monkey and then implanting a kidney from aclass II matched class I and minor antigen mismatched donor monkey intothe recipient. Tolerance to the donor kidney (as indicated by prolongedacceptance of the graft) is indicative that the putative agent is, atthe dosage tested, a help reducing agent.

“Help reduction”, as used herein, means the reduction of T cell help bythe inhibition of the release of at least one cytokine, e.g., any ofIL-2, IL4, IL-6; gamma interferon, or TNF, from T cells of the recipientat the time of the first exposure to an antigen to which tolerance isdesired. The inhibition induced in a recipient's T cell secretion of acytokine must be sufficient such that the recipient is tolerized to anantigen which is administered during the reduction of help. Although notbeing bound by theory, it is believed that the level of reduction is onewhich substantially eliminates the initial burst of IL-2 whichaccompanies the first recognition of a foreign antigen but which doesnot eliminate all mature T cells, which cells may be important ineducating and producing tolerance.

“Hematopoietic space”, as used herein, refers to a condition created inthe bone marrow which promotes engraftment of administered stem cells.The most common way of creating hematopoietic space is by irradiation ofthe bone marrow with whole body irradiation.

“Hematopoietic stem cell”, as used herein, refers to a cell, e.g., abone marrow cell, or a fetal liver or spleen cell, which is capable ofdeveloping into all myeloid and lymphoid lineages and by virtue of beingable to self-renew can provide long term hematopoietic reconstitution.Purified preparations of hematopoietic cells or preparations, such asbone marrow, which include other cell types, can be used in methods ofthe invention. Although not wishing to be bound by theory, it isbelieved that the hematopoietic stem cells home to a site in therecipient mammal. The preparation should include immature cells, i.e.,undifferentiated hematopoietic stem cells; these desired cells can beseparated out of a preparation or a complex preparation can beadministered. E.g., in the case of bone marrow stem cells, the desiredprimitive cells can be separated out of a preparation or a complex bonemarrow sample including such cells can be used. Hematopoietic stem cellscan be from fetal, neonatal, immature or mature animals. Stem cellsderived from the cord blood of the recipient or the donor can be used inmethods of the invention. See U.S. Pat. No. 5,192,553, herebyincorporated by reference, and U.S. Pat. No. 5,004,681, herebyincorporated by reference.

“Immunosuppressive agent capable of inactivating thymic or lymph node Tcells”, as used herein, is an agent, e.g., a chemical agent, e.g., adrug, which, when administered at an appropriate dosage, results in theinactivation of thymic or lymph node T cells. Examples of such agentsare cyclosporine, FK-506, and rapamycin. Anti-T cell antibodies can alsobe used. An agent should be administered in sufficient dose to result insignificant inactivation of thymic or lymph node T cells which are notinactivated by administration of an anti-T cell antibody, e.g., ananti-ATG preparation. Putative agents, and useful concentrationsthereof, can be prescreened by in vitro or in vivo tests, e.g., byadministering the putative agent to a test animal, removing a sample ofthymus or lymph node tissue, and testing for the presence of active Tcells in an in vitro or in vivo assay. Such prescreened putative agentscan then be further tested in transplant assays.

“Thymic or lymph node or thymocytes or T cell”, as used herein, refersto thymocytes or T cells which are resistant to inactivation bytraditional methods of T cell inactivation, e.g., inactivation by asingle intravenous administration of anti-T cell antibodies, e.g.,anti-bodies, e.g., ATG preparation.

“Thymic irradiation”, as used herein, refers to a treatment in which atleast half, and preferably at least 75, 90, or 95% of the administeredirradiation is targeted to the thymus. Whole body irradiation, even ifthe thymus is irradiated in the process of delivering the whole bodyirradiation, is not considered thymic irradiation.

“MHC antigen”, as used herein, refers to a protein product of one ormore MHC genes; the term includes fragments or analogs of products ofMHC genes which can evoke an immune response in a recipient organism.Examples of MHC antigens include the products (and fragments or analogsthereof) of the human MHC genes, i.e., the HLA genes. MHC antigens inswine, e.g., miniature swine, include the products (and fragments andanalogs thereof) of the SLA genes, e.g., the DRB gene.

“Miniature swine”, as used herein, refers to a wholly or partiallyinbred pig.

“Hematopoietic space-creating irradiation”, as used herein, refers toirradiation directed to the hematopoietic tissue, i.e., to tissue inwhich stem cells are found, e.g., the bone marrow. It is of sufficientintensity to kill or inactivate a substantial number of hematopoieticcells. It is often given as whole body irradiation.

“Thymic space” as used herein, is a state created by a treatment thatfacilitates the migration to and/or development in the thymus of donorhematopoietic cells of a type which can delete or inactivate hostthymocytes that recognize donor antigens. It is believed that the effectis mediated by elimination of host cells in the thymus.

“Short course of a help reducing agent”, as used herein, means atransitory non-chronic course of treatment. The treatment should-beginbefore or at about the time of transplantation of the graft.Alternatively, the treatment can begin before or at about the time ofthe recipient's first exposure to donor antigens. Optimally, thetreatment lasts for a time which is approximately equal to or less thanthe period required for mature T cells of the recipient species toinitiate rejection of an antigen after first being stimulated by theantigen. The duration of the treatment can be extended to a timeapproximately equal to or less than two, three, four, five, or tentimes, the period required for a mature T cell of the recipient speciesto initiate rejection of an antigen after first being stimulated by theantigen. The duration will usually be at least equal to the timerequired for mature T cells of the recipient species to initiaterejection of an antigen after first being stimulated by the antigen. Inpigs and monkeys, about 12 days of treatment is sufficient. Experimentswith cyclosporine A (10 mg/kg) in pigs show that 6 days is notsufficient. Other experiments in monkeys show that IL-2 administered onday 8, 9, or 10 of cyclosporine A treatment will result in rejection ofthe transplanted tissue. Thus, 8, 9, or 10 days is probably notsufficient in pigs. In monkeys, a dose of 10 mg/kg cyclosporine with ablood level of about 500-1,000 ng/ml is sufficient to induce toleranceto class II matched class I and minor antigen mismatched kidneys. Thesame blood level, 500-1,000 ng/ml, is sufficient to induce tolerance inpigs. Long-term administration of 5 mg/kg prevents rejection (by longterm immune suppression) but does not result in tolerance.

“Short course of a immunosuppressive agent”, as used herein, means atransitory non-chronic course of treatment. The treatment should beginbefore or at about the time the treatment to induce tolerance is begun,e.g., at about the time, xenogeneic, allogeneic, genetically engineeredsyngeneic, or genetically engineered autologous stem cells areintroduced into the recipient. e.g., the short course can begin on theday the treatment to induce tolerance is begun, e.g., on the day,xenogeneic, allogeneic, genetically engineered syngeneic, or geneticallyengineered autologous stem cells are introduced into the recipient orthe short course can begin within 1, 2, 4, 6, 8, or 10 days before orafter the treatment to induce tolerance is begun, e.g., within 1, 2, 4,6, 8, or 10 days before or after xenogeneic, allogeneic, geneticallyengineered syngeneic, or genetically engineered autologous stem cellsare introduced into the recipient. The short course can last for: aperiod equal to or less than about 8-12 days, preferably about 10 days,or a time which is approximately equal to or is less than two, three,four, five, or ten times the 8-12 or 10 day period. Optimally, the shortcourse lasts about 30 days. The dosage should be sufficient to maintaina blood level sufficient to inactivate thymic or lymph node T cells. Adosage of approximately 15 mg/kg/day has been found to be effective inprimates.

“Stromal tissue”, as used herein, refers to the supporting tissue ormatrix of an organ, as distinguished from its functional elements orparenchyma.

“Tolerance”, as used herein, refers to an inhibition of a graftrecipient's immune response which would otherwise occur, e.g., inresponse to the introduction of a nonself MHC antigen into therecipient. Tolerance can involve humoral, cellular, or both humoral andcellular responses. Tolerance, as used herein, refers not only tocomplete immunologic tolerance to an antigen, but to partial immunologictolerance, i.e., a degree of tolerance to an antigen which is greaterthan what would be seen if a method of the invention were not employed.Tolerance, as used herein, refers to a donor antigen-specific inhibitionof the immune system as opposed to the broad spectrum inhibition of theimmune system seen with immunosuppressants.

Methods of the invention minimize or eliminate the need forhematopoietic space-creating treatment, e.g., irradiation, in manymethods of tolerance induction.

In methods of the invention, the creation of thymic space, e.g., bythymic irradiation, the inactivation of recipient peripheral T cells andthymocytes, and the administration of a sufficiently large number ofxenogeneic or allogeneic donor stem cells allows the induction oftolerance without subjection the recipient to WBI. The induction ofthymic space can reduce the level of donor reactive thymocytes butadditional steps (described herein) can be added to further diminishdonor thymocyte reactivity.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION

The drawings will first be briefly described.

Drawings

FIG. 1 is a depiction of multilineage analysis of donor repopulation inanimals administered either one injection (----) or five injections(______) of BMC.

FIG. 2 (left panel) is a graph of long-term donor monocyte (◯),granulocyte (▪), and B cell (solid triangle) repopulation in WBC ofstable chimeras among B6 mice receiving anti-CD4 and CD8 mAbs on days−5, −1 and 7, 6 Gy TI on day 0, with 174×10⁶B10.A BMC over five days,from day 0 through 4 (n=7). Standard deviations are shown on each datapoint. The right panel shows that Mean ±SD percentages of total CD4 (Δ)and CD8 (▪) cells of donor origin in WBC of the same mice shown in theleft panel.

FIG. 3 is a depiction of CML responses of spleen cells from stable mixedchimeras. B6 mice treated with anti-CD4 and CD8 mAbs on days −5, −1 and7, TI on day 0, and high-dose B10.A BMC(174×10⁶ cells over days 0through 4) were analyzed 25 to 29 weeks post-BMT. CML reactivity tohost-type (top left panel), donor-type (top right panel), and thirdparty (bottom panel) stimulators and targets is shown for: (□) a mixedchimera; (*) a non-BMT control/(◯) a normal B6 mouse and; () a normalB10.A mouse. Percent specific lysis was calculated using the followingformula: 100%×(Experimental ⁵¹Cr release−Spontaneous ⁵¹Crrelease)/(Maximum ⁵¹Cr release−Spontaneous ⁵¹Cr release). The chart atthe bottom of the figure shows maximum % specific lysis obtained forthree additional chimeras.

FIG. 4 is a graph showing the specific acceptance of donor-specific skingrafts in B6 recipients of allogeneic B10.A BMT (174×10⁶ days 0 through4) after treatment with anti-CD4 and CD8 mAbs on days −5, −1 and 7, with7 Gy of TI on day 0. Left panel: Survival of donor-type skin onsimilarly-treated non-BMT control mice (______) and BMT recipients(---). Right panel: Survival of third party (SJL/J) skin on the samegroups of mice. Grafting was performed seven weeks post-BMT.

FIG. 5 is a depiction of the specific deletion of Vβ5+ and Vβ11+ cellsamong CD4+ spleen cells and among mature (class I^(high)) host-type(K^(bhigh)) thymocytes of chimeras sacrificed 24 to 29 weeks post BMT.For b10.A control mice, gated H-2D^(bhigh) thymocytes were analyzedinstead. B6 mice received 174×10⁶ B10.A BMC over days 0-4 afterconditioning with anti-CD4 and -CD8 mAbs and 7 Gy TI. FCM analysis wasperformed on 10⁴ gated cells for each population of interest. The totalnumber of TCRaβ^(high) cells in the same gate was also determined, andthe results obtained for each individual Vβ were corrected by dividingby the fraction of TCRaβ^(high) cells. Results are presented for stablechimeras (n=6). * denotes P<0.05; ***, P<0.005; ****, P<0.0005; *****,P<0.00005 compared to simultaneous, similarly-treated non-BMT controls(n=4).

OVERVIEW

The invention provides several methods of inducing tolerance to foreignantigens, e.g., to antigens on allogeneic or xenogeneic tissue or organgrafts. These methods can be used individually or in combination.

Section I below presents animal trials in which it is shown thatengraftment, mixed chimerism, and tolerance can be induced without theneed for hematopoietic space-creating irradiation.

Section II below describes sources of cells for transplantation.

Section III below discusses implantation of bone marrow cells to inducetolerance to MHC disparity.

I. EXAMPLE 1 The Effect of Thymic Irradiation on Syngeneic EngraftmentUsing High Doses of Donor Bone Marrow

Animals

Female C57BL/6NCR (B6; H-2b. Ly-5.2) and female Ly-5 congenic B6.Ly-5.2(Ly-5.1) mice were obtained from the Frederick Cancer Research Facility(Frederick, Md.). Ly-5 alleles are described according to thenomenclature of Morse et al. (1987. Immunogenetics 25:71). All mice werehoused in sterilized microisolator cages in which they receivedautoclaved food and autoclaved acidified drinking water. Recipients wereage-matched and were used at 12 to 16 weeks of age.

BMT

C57BL/6NCR (B6; H-2b. Ly-5.2) mice received 100 ml each of ascitescontaining anti-CD4 mAb GK1.5 and anti-CD8 mAb 2.43 intraperitoneally ondays −6 and −1 and +7. This volume of ascites contained 1-2 mg of GK1.5and 1.25-1.5 mg of 2.43 respectively, as measured by rat IgG2b-specificELISA. Animals were irradiated with either 0, 3.5 or 7 Gy thymicirradiation on day 0. One series of mice was treated with one dose of200 million bone marrow cells (BMC). A second series of mice weretreated, starting on day 0 and repeated daily for a total of five days,with forty million (total 200 million cells) BMC from Ly-5 congenicB6.Ly-5.2 (Ly-5.1) mice were administered intravenously. BM cells (BMCs,200×10⁶) were obtained from the tibiae and femora of sexmatched B6,Ly-5.2 donors aged 6 to 14 weeks. T cell depletion was performed asdescribed in Sykes et al. (1990. PNAS, 87: 5633-5637) using anti-CD4 andCD8 mAbs and rabbit complement.

Cell Counts

Heparinized peripheral blood was analyzed on an Automated Cell Counter(System 9000; Serono-Baker Diagnostics Inc. Allentown, Pa.).

Phenotyning

Phenotyping was performed at various times beginning 2 weeks after BMT.Animals were tail bled and white blood cells (WBCs) were prepared byhypotonic shock. Suspensions of spleen cells, thymocytes. BMCs, and BMcolonies were also analyzed. Staining with both donor-specific andrecipient-specific mAB was performed on each chimera and control animal.Cells were incubated with 20 mL undiluted culture supernatant of A20-1.7(anti-Ly-5.1 mAb; mouse IgG2a) or 104-2.1 (anti-Ly-5.2 mAB; mouse IgG2a)(hybridomas kindly provided by Dr. S. Kimura, Sloan Kettering CancerInstitute. New York, N.Y.) for 30 minutes at 4° C. and then washedtwice. To block nonspecific FcgR binding of labeled antibodies, 10 mLundiluted culture supernatant of 2.4G2 (rat antimouse FcgR mAb) wasadded to the first incubation. Cell-bound mAbs were detected withfluorescein isothiocyanate (FlTC)-conjugated rat antimouse IgG2a mAb(Zymed laboratories. Inc. Mundelein, Ill.), which was incubated for 30minutes at 4° C. followed by two washes and analysis on an FACScan(Becton Dickinson, Mountain View, Calif.). In all experiments, thepercentage of cells staining with each mAb was determined from one-colorfluorescence histograms and comparison with those obtained from normaldonor and host-type animals, which were used as positive and negativecontrols. The percentage of cells considered positive after stainingwith an mAb was determined using a cutoff chosen as the fluorescencelevel at the beginning of the positive peak for the positive controlstrain, and by subtracting the percentage of cells stained with anirrelevant mAb (nonreactive IgG2a mAb HOPC1 plus FITC-conjugatedantimouse IgG2a mAb). The relative percent staining of a chimera withmAb was calculated using the formula: 100% ×(net chimera percentpositive)−(net negative control percent positive)/(net positive controlpercent positive)−(net negative control percent positive), in which netpercent positive refers to the percentage obtained after subtraction ofstaining with HOPC1, and positive and negative controls were cells fromappropriate normal Ly-5.1 + and Ly-5.2+ mice. For test cell populationsin which staining with an anti-Ly-5 mAb was less than that of thenegative control and the calculated percent chimerism was therefore lessthan 0, the values are reported as 0. Using this method of calculation,less than 0.1% contaminating Ly-5.1+ cells could be detected inartificial Ly-5.2 (99.9%)/Ly-5.1 (0.1%) mixtures. However, a visiblepositive peak was not detectable in artificial mixtures containing 0.1;or fewer Ly-5.1+ cells, but was visible with 1% contaminating Ly-5.1+cells (data not shown). All hematopoietic lineages stained strongly withanti-Ly-5 mAb. By using forward and 90° light scatter (FSC and SSCrespectively) dot plots lymphocyte (FSC- and SSC-low population),granulocyte (SSC-high population) and monocyte (FSC-high but SSC-lowpopulation) populations were gated, and chimerism was determinedseparately for each population. All SSC-high cells in the granulocytegate stained with FITC-conjugated antimouse granulocyte mAb (Gr-1). Deadcells were excluded by gating out low FSC/high propidiumiodide-retaining cells.

Engraftment Without Thymic Irradiation

Two groups of animals were administered either one injection of 20×10⁷BMC or five injections on a daily basis of 40×10⁶ BMC. Multilineageanalysis of donor repopulation showed that all lineages showed 10-25%long-term chimerism which remained stable for at least 30 weeks (FIG.1). Thus, when multiple high doses of bone marrow cells are injectedinto the recipient mouse stable engraftment of hematopoietic stem cellscan occur.

Engraftment With Thymic Irradiation

Significantly high levels of engraftment were observed in the CD4 T cellpopulation when the mice were pretreated with thymic irradiation Table1, increasing from approximately 0-10% to between 20-60%. Repopulationof the monocyte lineage was increased from a level of approximately 20%to between 30 to 40%. This is in agreement with the results previouslyshown that 3.5 Gy WBI is sufficient to enable stable engraftment in thesyngeneic setting. These results indicate that while engraftment can beachieved in the absence of either WBI or TI a relatively low dose ofthymic irradiation (3.5 Gy) enables a higher level of syngeneicengraftment to be obtained.

TABLE 1 The effect of Thymic Irradiation on Syngeneic Engraftment %Engraftment at Day 30 Thymic Irradiation CD4+ Monocytes 0  ND* 20.8 11.518.2 ND 25.3 ND 19.4 3.5 Gy 31.0 31.4 36.6 31.5 64.3 28.8 28.4 29.6 7.0Gy 37.0 39.0 71.3 36.7 41.2 41.5 *Not detectable

EXAMPLE 2 Induction of High Levels of Allogeneic HematopoieticReconstitution and Donor-specific Tolerance without MyelosuppressiveConditioning

Pluripotent hematopoietic stem cells (PHSC) engraft in unconditionedrecipients given high doses of syngeneic or Ly5 congenic marrow.Allogeneic PHSC engraftment was achieved by administering a high dose(200×10⁶) of fully MHC-mismatched B10.A (H-2^(a)) BMC to B6 (H-2^(b))recipients. The recipients were conditioned only with depleting anti-CD4and anti-CD8 mAbs on days −5, −1 and 7. Initial chimerism was achievedamong peripheral blood lymphocytes, monocytes and granulocytes, withpeak levels of 15-33% donor cells at four to six weeks post-BMT.However, initial T cell chimerism was low (<10%), and multilineagechimerism tended to decline with time. Table 2 shows the low levels ofchimerism in spleen, marrow and thymus of animals sacrificed 12 to 25weeks after BTM.

In order to optimize engraftment thymic space was induced by theadministration of thymic irradiation (TI). B6 mice received anti-T cellmAbs as above, 7 Gy TI on day 0, and a total of 174×10⁶ fullyMHC-mismatched B10.A BMC on days 0 through 4. In seven of ten animals,donor cells constituted a high proportion of WBC monocytes,granulocytes, B cells and CD4 and CD8 cells at all times (FIG. 2). Inseven animals, the level of initial donor CD4 T cell was similar to thatof other lineages, and chimerism in all lineages was stable throughoutthe six-month follow-up period (FIG. 2). However, in three animals,despite initially high levels of chimerism, donor representationdeclined in some or all lineages over time (data not shown).

Recipients of the TI-containing high-dose allogeneic BMT regimen weresacrificed 24 to 29 weeks following BMT, and chimerism was evaluated inother tissues. Most stable WBC chimeras showed substantial chimerismamong BMC, splenic B and T cells, and class I^(high), mature thymocytes(Table 2). These thymi also contained donor class I^(low), immaturethymocytes. Unlike stable chimeras, thymi from the three “unstable”chimeras (Table 2) contained few donor-derived class I^(high) cells, andshowed variable splenic T and B cell and BMC chimerism (Table 2).

The stable, high-level, multilineage chimerism observed in most micedemonstrates that substantial allogenic PHSC engraftment can be achievedwithout WBI in mice receiving T cell-depleting mAbs, 7 Gy TI, andhigh-dose allogeneic marrow. Donor representation was similar to thatobserved in similarly-treated recipients of Ly5 congenic marrow,indicating that immunologic alloresistance was completely overcome.These results are consistent with our previous studies demonstratingthat the minimal barrier posed by recipient NK cells to allogeneic PHSCengraftment could be readily overcome by administering additional BMC,suggesting that NK cell-mediated resistance is saturable. Theobservation that greater numbers of allogeneic than syngeneic purifiedstem cells are required to rescue lethally irradiated mice may reflect afailure to completely overcome T cell-mediated alloresistance. Thepresence of “facilitating” cell populations in whole marrow inocula isunlikely to have affected our results, since such cell types havegenerally been reported to express CD4 or CD8, and donor CD4 and CD8cells are depleted by mAbs present in the circulation at the time ofBMT.

TABLE 2 Chimerism in B6 recipients of high-dose allogeneic B10.A marrow,anti-CD4 and CD8 mAbs, and 7 Gy TI sacrificed 24 to 29 week post-BMT.Percentage of donor cells Thymus^(a) Spleen Donor Donor Animal BMC CD4CD8 B Cells Class I^(hi) Class II^(hi) B10.A (n = 4) 92.18 ± 5    99.77± 0.1  99.37 ± 0.6  99.76 ± 0.2  91.48 ± 6    + No BMT^(b) (n = 4) 0.06± 0.06 0 ± 0 0 ± 0 0.63 ± 0.1 0.11 ± 0.2  − Expt. 1: BMT^(c) (200 × 10⁶B10.A BMC days 0-4), without TI^(d) 1  ND^(a) 0 0.07 0.07 0 ND 2 ND NDND 0.05 0 ND 3 6.98 0.47 0.25 11.29 ND ND Expt. 2: BMT^(c) (174 × 10⁶B10.A BMC days 0-4), with TI Stable 1 36.99 39.17 38.38 63.13 38.39 +Chimeras: 2 12.38 31.14 26.71 46.32 12.27  ±^(f) 3 21.90 6.32 9.86 27.6921.83 + 4 45.40 35.12 31.74 55.71 24.46 + 5 36.03 2.73 10.74 2.2328.23 + 6 46.63 35.24 31.58 56.89 34.20 + Unstable 1 0.33 9.10 4.15 7.434.95 − Chimeras: 2 12.00 19.94 17.45 40.02 6.65 + 3 0.55 2.61 9.74 2.316.83 − ^(a)Percentage of total class I^(high), TCRαβ^(high) thymocytesthat were of donor type. ^(b)These controls were B6 mice that receivedidentical treatment simultaneously with the BMT recipients in Expt. 2,but which did not receive BMT. They received anti-CD4 and CD8 mAbs ondays -5, -1 and 7 and 7 Gy TI on day 0. ^(c)All animals receivedanti-CD4 and CD8 mAbs on days -5, -1 and 7.

The mice were evaluated for myelosuppression. Complete blood counts weredetermined on days −1, 1, 3, 6, 8, 10, 13, and 20 for animals receivingTI on day 0 with or without mAb treatments, without BMT. In recipientsof TI±mAbs, average WBC counts reached a nadir of 0.3,000/μl on day 1,and returned to normal by day 8. The lowest level reached in anyindividual mouse was 2,600/μl. Neither platelet counts nor hemoglobinconcentrations decreased significantly at any time in any group. Allanimals survived with no clinical toxicity. Therefore, host conditioningwith mAbs/TI does not cause clinically significant myelosuppression ortoxicity, yet permits PGHSC engraftment from high-dose allogeneicmarrow.

High-does allogeneic BMT recipients showed no detectable weight loss orother clinical signs of acute or chronic GVHD. The timing of T cellrecovery was similar in animals receiving mAbs/TI conditioning with orwithout high-dose allogeneic BMT, and thymic and splenic cell yieldswere similar in both groups. The freedom of the recipients from clinicalstigmata or lymphoid atrophy associated with GVHD probably reflectsdepletion of mature T cells in donor marrow by mabs that are stillpresent in the serum at the time of BMT.

To evaluate tolerance, mixed lympyhocyte reactions (MLR) and cellmediated lympholysis (CML) studies were performed in BMT recipients andsimultaneous, similarly-treated non-MBT controls 24 to 29 weekspost-conditioning. All four animals with stable multilineage chimerismshowed specific CML tolerance to donor and host, with similar anti-thirdparty responses as non-BMT control mice (FIG. 3). The latter groupshowed similar anti-B10.A responses to those of untreated B6 mice (FIG.3). Four of six stable chimeras showed donor-specific MLRunresponsiveness, while two animals were generally hyporesponsive. Incontrast, all four non-BMT controls showed similar anti-B10.A responsesto those of B6 mice (P<0.01 compared to BMT mice). Overall, the resultsdemonstrate donor-specific CML and MLR tolerance in mice receivinghigh-dose allogeneic BMT with non-myelosuppressive conditioning.

Of two “unstable chimeras” in Table 2, one showed donor-specific CMLunresponsiveness and another showed generalized unresponsiveness in CML.Two of the three unstable chimeras showed donor-specific MLRunresponsiveness, while the third showed generalized unresponsiveness(data now shown). The robust responses observed for conditioned non-BMTcontrols rules out the conditioning regimen itself as the cause of thishyporesponsiveness, and its presence in unstable chimeras, and the lackof evidence for GVHD makes GVH-associated immunodeficiency an unlikelyexplanation. Cross-reactivity of third party antigens with donorantigens to which the animals were tolerant is the most likelyexplanation for the weak third party responses in some BMT recipients.

Since some unstable as well as stable chimeras showed donor-specificunresponsiveness in vitro, the decline in chimerism in unstable chimerasmay be a more sensitive indicator of incomplete tolerance than MLR orCML. Alternatively, declining chimerism may reflect non-immunologicmechanisms, such as poor PHSC engraftment. To distinguish between thesepossibilities, tolerance was evaluated by the most stringent test, skingrafting. All stable chimeras permanently accepted donor skin grafts,but rapidly rejected third party grafts, thereby demonstratingdonor-specific tolerance (FIG. 4). One unstable chimera in which thedeclining chimerism was confined to the T cell lineage (“unstablechimera” #2, Table 2) also accepted donor skin. The other two unstablechimeras chronically rejected donor-type skin by days 105 and 48,respectively. In stable chimeras, repeat donor skin grafted 31 weekspost-BMT (and the original grafts) remained in perfect condition untilthe time of sacrifice three to eight weeks later. Thus, by the moststringent criterion of skin grafting, these animals showed permanent,donor-specific tolerance.

Vβ usage was analysed to examine the mechanism of tolerance in chimerasprepared with high-dose allogeneic marrow. The donor strain, B10.A,expresses I-E, which is required to present Mtv-derived superantigensencoded in the B6/B10 background genome. Developing thymocytes whose TCRcontain Vβ11 or Vβ5, which bind to these superantigens, are deleted inB10.A mice, but not in B6 (H-2^(b)) mice, which do not express I-E (FIG.5). Vβ11+ and Vβ5+ mature host thymocytes (gated H-2K^(b high) cells)and peripheral CD4+ cells were enumerated. Long-term stable chimerasshowed deletion of Vβ5 and Vβ11 CD4+ cells among PBL, splenocytes andmature B6 thymocytes, similar to normal B10.A donors. These Vβ were notdeleted in non-BMT controls. Percentages of control Vβ8.½ cells werenormal in all groups (FIG. 5; PBL data not shown). The “unstablechimeras” in Table 2 showed less complete deletion of Vβ5+ and Vβ11+host-type thymocytes (1.4-4.2% Vβ5, 1.1-5.1% Vβ11) and of CD4 spleencells and PBL (not shown) than did stable chimeras. Thus, completedeletion of Vβ that recognize donor I-E plus superantigens correlatedwith the presence of donor-specific skin graft tolerance and permanent,stable mixed chimerism, suggesting that intrathymic deletion was amechanism of tolerance.

Since hematopoietic cells can efficiently induce clonal deletion in thethymus, we looked for donor I-E in recipient thymi usingimmunohistochemistry. Donor I-E+ cells were clearly detectable 24 to 29weeks post-BMT in thymi of most stable chimeras (Table 2). In contrast,two of three unstable chimeras did not contain detectable donor I-E+cells in their thymi (Table 2). Overall, these results demonstrate acorrelation between the long-term intrathymic presence of donor-derivedclass II+ cells and complete deletion of Vβ that recognize superantigenspresented by donor MHC molecules. Host class II+ cells were distributednormally in thymi of all recipients. Thymic irradiation was essential tothe optimization of stable chimerism (Table 1) and permanent skin grafttolerance. While 7 Gy TI was not significantly myelosuppressive, itpermitted high levels PHSC engraftment and permanent, deletional,donor-specific tolerance.

Peripheral chimerism may be achieved through the high doses of bonemarrow without the need for whole irradiation. However, to achievecentral deletional tolerance it is best to create space in the thymus inorder to allow high levels of intrathymic chimerism to develop. This canbe achieved by irradiation or by the use of multiple administrations ofanti-T cell antibodies or with drugs that deplete the thymus. A level ofthymic irradiation between 3 and 7 Gy may be appropriate.

MATERIALS AND METHODS

Animals

Female C57BL/6 (B6:H-2^(b)), B10.A (B10.A:H-2^(a), K^(k), I-A^(k),I-E^(k), D^(d)), BALB/c (H-2^(d)), SJL (H-2^(a)) and A.SW (H-2^(a)) micewere purchased from Frederick Cancer Research Center, Frederick, Md., orfrom The Jackson Laboratory, Bar Harbor, Me. Mice were maintained in aspecific pathogen-free microisolator environment.

Conditioning and BMT

Age-matched (7 to 14 weeks old) female B6 recipient mice received 2 mgand 1.4 mg of rat IgG_(2b) anti-mouse CD4 mAb GK1.5 (Dialynas et al., J.Immunol. 131:2445-2451 (1983), hereby incorporated by reference) andanti-mouse CD8 mAb 2.43 (Sarmiento J. Immunol. 125:2665 (1980), herebyincorporated by reference), respectively, intraperitoneally (i.p.) ondays −5, −1 and 7 with respect to BMT. 7 Gy selective thymic irradiationwas given on day 0 (Sharabi et al., J. Exp. Med. 169:493-502 (1989),hereby incorporated by reference). 35-40×10⁶ untreated BMC from B10.Amice were administered daily on each of days 0 through 4, for a total offive injections (total 174-200×10⁶ BMC).

Mabs

Non-specific FcgR binding was blocked anti-mouse FcgR mAb 2.4G2 (Shermanet al., Immunogenetics 12:183-189 (1981), hereby incorporated byreference). FITC-conjugated mAbs included anti-CD4 (Pharmingen, SanDiego, Calif.), anti-CD8 (Caltag, San Francisco, Calif. and Pharmingen),anti-MAC1 (Caltage) and rat anti-mouse IghM (ymed) mAbs, as well asanti-TCRaβ, -Vb5, -Vb11 and -Vb8.½ mAbs purchased from Pharmingen.Negative control mAb HOPC1-FITC, with no reactivity to mouse cells, wasprepared in our laboratory. Biotinylated anti-H-2D^(d) mAb 34-2-12(Ozato et al., Transplantation 34:113-120 (1982), hereby incorporated byreference), anti-H-2K^(b) mAb 5F1 (Sherman et al., Immunogenetics12:183-189 (1981), hereby incorporated by reference) and control mAbHOPC1 were developed with phycoerythrin-streptavidin (PEA).Phycoerythrin-conjugated anti CD4 mAb (Pharmingen, San Diego, Calif.)and non-specific rat IgG2a (negative control) were purchased fromPharmingen.

Flow Cytometric (FCM) Analysis of Multilineage Chimerism

Allogeneic reconstitution of various lineages in WBC, spleen marrow andthymus was evaluated by Two-clor FCM. Forward angle and 90 degree lightscatter properties were used to distinguish lymphocytes, granulocytesand monocytes in WBC, as described. Two-color FCM was utilized todistinguish donor and host cells of particular lineages, an thepercentage of donor cells was calculated as described (Lee et al.,Transplantation 61:125-132 (1996); and Tomita et al., J. Immunol.153:1087-1098 (1994), both hereby incorporated by reference), bysubtracting control staining from quadrants containing donor and hostcells of a particular phenotype, and dividing the net percentage ofdonor cells by the total net percentage of donor plus host cells of thatphenotype. Dead cells were excluded by gating out low FSC/high propidiumiodide-retaining cells. For analysis of T cell receptor (TCR) Vβfamilies, 10⁴ gated CD4+ T cells (PBL and spleen) or 10⁴ gated H-2 classI^(high) thymocytes were analyzed as described (Tomita et al., J.Immunol. 153:1087-1098 (1994), hereby incorporated by reference). ClassI^(high) thymocytes include mainly mature, single positive T cells(Scollay et al., J. Immunol. 124:2845 (1980), hereby incorporated byreference).

Mixed Lymphocyte Reactions (MLR)

Splenocytes were cultured in triplicate wells containing 4×10⁵responders with 4×10⁵ stimulators (30 Gy) in RPMI 1640 mediumsupplemented with 15% (vol/vol) controlled processed serum replacement(CPSR-2; Sigma), 4% nutrient mixture (7.3 mg/ml L-glutamine, 4×non-essential amino acids (Gibco), 2.75 mg/ml sodium pyruvate, 250 U/mlpenicillin and 250 mg/ml streptomycin), 1% Hepes buffer, and 10 mM2-mercaptoethanol at 37° C. in 5% CO2 for three to four days before theywere pulsed with ³H-labeled thymidine and harvested 18 hours later.Stimulation index (S.I.) was calculated by comparing anti-donor andanti-third party responses with anti-host responses, which were similarto background counts (i.e., cpm with no stimulator cell population).

Cell-mediated Lympholysis (CML) Reactions

CML reactions were performed as described (Sykes et al. J. Immunol.140:2903-2911 (1988), hereby incorporated by reference), except that8×10⁵ responders were cultured with 8×10⁵ stimulators (30 Gy irradiated)in each well, and 8000⁵¹Cr-labeled 48-hour concanavalin A lymphoblastswere added on day 5.

Skin Grafting

Donor-type and third party (SJL) full thickness tail skin grafts wereimplanted as described (Sharabi et al., J. Exp. Med. 169:493-502 (1989),hereby incorporated by reference). Grafts were defined as accepted ifthey were in perfect condition, with tail hairs and scales, and wereconsidered rejected at the time of complete sloughing or when theyformed a dry scab.

Immunohistochemistry

Four micron sections were prepared from frozen thymus tissue and stainedwith mAbs ISCR3 (Watanabe et al., Transplantation 36:712-718 (1983),hereby incorporated by reference).(mouse IgG_(2b) anti I-E), 25-9-17(mouse IgG_(2a) anti-I-A^(b)) (Ozato et al., J. Immunol. 126:317-321(1981), hereby incorporated by reference), HOPC-1 (mouse IgG_(2a)isotope control) or 74-11-10 (mouse IgG_(2b) isotope control), developedwith biotinylated rat anti-mouse IgG_(2a) or anti-IgG_(2b) (Pharmingen),streptavidin-horseradish peroxidates and substrate, as described (Tomitaet al., J. Immunol. 153:1087-1098 (1994), hereby incorporated byreference). Stained sections were analyzed by an observer who wasunaware of the animal from which the tissue had been obtained.

Statistical Analysis

Statistical significance was determined using Student's t-test forcomparison of means. A p value of less than 0.05 was considered to bestatistically significant.

II. Sources of Cells for Allogeneic Stem Cell Transplantation

A living human donor can provide about 7.5×10⁸ bone marrow cells/kg.Methods of the invention can include the administration of at least 2 or3 times this number (per kg) and preferably at least 10, 15, or 20 timesthis number. The requisite numbers of bone marrow cells can be providedby the ex vivo expansion or amplification of human stem cells. Ex vivoexpansion is reviewed in Emerson, 1996, Blood 87:3082, herebyincorporated by reference. Methods of ex vivo expansion are described inmore detail in Petzer et al., 1996, Proc. Natl. Acad. Sci. USA 93:1470;Zundstra et al., 1994, BioTechnology 12:909; and WO 95 11692 Davis etal., all of which are hereby incorporated by reference. Sources ofhematopoietic stem cells include bone marrow cells, mobilized peripheralblood cells, and when available cord blood cells.

Sources of Cells for Xenogeneic Stem Cell Transplantation

In the case of inbred donor animals, e.g., inbred miniature swine, verylarge numbers of stem cells are available, as the number which can besupplied is not limited by the number which can be harvested from asingle donor.

In the case where the recipient is a primate, e.g., a human, and thedonor is a swine, e.g., a miniature swine, 7.5×10⁹ or more, andpreferably, between 7.5×10⁹ and 15×10¹⁰, swine bone marrow cells/kg canbe administered, though this will vary with factors such as theintensity of the preparative regimen and the health of the individualrecipient. As discussed herein, these cells can be provided in more thanone administrations.

Determination of the Number of Swine Bone Marrow Cells Needed to InduceTolerance

The following system can be used to determine or refine the number ofswine cells needed to engraft and induce tolerance in a swine—primatemodel. Various doses of donor cells are administered to cynomolgusmonkeys and the number of cells required for the establishment ofchimerism and induction of tolerance determined by the assays described.Time zero is defined as the moment that the arterial and venous cannulasof the recipient are connected to the liver to be perfused.

Thymic space is induced by administering 700 rad of thymic irradiationbetween days −l and −8. WBI is not administered.

On day −1 a commercial preparation (Upjohn) of horse anti-humananti-thymocyte globulin (ATG) is injected into the recipient. Therecipient is anesthetized, an IV catheter is inserted into therecipient, and 6 ml of heparinized whole blood are removed beforeinfusion. The ATG preparation is then injected (50 mg/kg) intravenously.Six ml samples of heparinized whole blood are drawn for testing at timepoints of 30 min., 24 hours and 48 hours. Blood samples are analyzed forthe effect of antibody treatment on natural killer cell activity(testing on K562 targets) and by FACS analysis for lymphocytesubpopulations, including CD4, CD8, CD3, CD11b, and CD16. If mature Tcells and NK cells are not eliminated, ATG can be re-administered atlater times in the procedure.

To remove natural antibodies from the recipient's circulation prior totransplantation, on day 0 an operative absorption of natural antibodies(nAB) is performed, using a miniature swine liver, as follows. At −90minutes the swine donor is anesthetized, And the liver prepared forremoval by standard operative procedures. At −60 minutes the recipientmonkey is anesthetized. A peripheral IV catheter is inserted, and a 6 mlsample of whole blood is drawn. Through mid-line incision, the abdominalaorta and the vena cava are isolated. Silastic cannulas containing sideports for blood sampling are inserted into the blood vessels.

At −30 minutes the liver is perfused in situ until it turns pale, andthen removed from the swine donor and placed into cold Ringers Lactate.The liver is kept cold until just prior to reperfusion in the monkey. Aliver biopsy is taken. At −10 minutes the liver is perfused with warmalbumin solution until the liver is warm (37 degrees).

At 0 time the arterial and venous cannulas of the recipient areconnected to the portal vein and vena cava of the donor liver andperfusion is begun. Liver biopsies are taken at 30 minutes and 60minutes, respectively. Samples of recipient blood are also drawn forserum at 30 minutes and 60 minutes respectively. At 60 minutes the liveris disconnected from the cannulas and the recipient's large bloodvessels are repaired. The liver, having served its function of absorbingharmful natural antibodies from the recipient monkey, is discarded.Additional blood samples for serum are drawn from the recipient at 2,24, and 48 hours. Organ perfusion can be replaced by perfusion of anα1-3 galactose linkage epitope-affinity matrix, e.g., in the form of anaffinity column, e.g., matrix bound linear B type VI carbohydrate.

Swine donor bone marrow cells are administered by intravenous injection.Bone marrow is harvested and injected intravenously as previouslydescribed (Pennington et al., 1988, Transplantation 45:21-26).7.5×10⁸/kg bone marrow cells are typically administered in regimenswhich include WBI. Initial trials to determine an appropriate number ofcells to be administered in a regimen which lacks WBI should begin witha range of doses from several times to 20 times that number. Multipleadministrations are desirable in the higher end of the dosage range.Swine cytokines can be administered to promote engraftment.

To follow chimerism, two color flow cytometry can be used. This assayuses monoclonal antibodies to distinguish between donor class I majorhistocompatibility antigens and leukocyte common antigens versusrecipient class I major histocompatibility antigens. Alternativelychimerism can be followed by PCR. Should natural antibodies be found torecur before tolerance is induced, and should these antibodies causedamage to the donor tissue, the protocol can be modified to permitsufficient time following BMT for humoral tolerance to be establishedprior to organ grafting. Tolerance to donor antigen can be followed bystandard methods, e.g., by MLR assays.

III. The Induction of Tolerance with Bone Marrow Transplantation

The following procedure was designed to lengthen the time an implantedorgan (a xenograft) survives in a xenogeneic host prior to rejection.The organ can be any organ, e.g., a liver, a kidney, a pancreas, or aheart. The method main strategies include one or more of the following:the elimination of natural antibodies, e.g., by contacting therecipient's blood with epitopes which react with donor-reactive naturalantibody; inactivation of host T cells; inactivation of host NK cells;transplantation of tolerance-inducing stem cells, e.g., bone marrow stemcells, optionally, the implantation of donor stromal tissue oradministration of donor cytokines; and the administration of thymicirradiation. The combination of a sufficiently large number ofadministered donor stem cells in combination with thymic irradiationsignificantly reduces or eliminates the need for WBI. The methodincludes any or all of these steps. Preferably they are carried out inthe following sequence.

First, a preparation of horse anti-human thymocyte globulin (ATG) isintravenously injected into the recipient. The antibody preparationeliminates mature T cells and natural killer cells. If not eliminated,mature T cells would promote rejection of both the bone marrowtransplant and, after sensitization, the xenograft itself. The ATGpreparation also eliminates natural killer (NK) cells. NK cells probablyhave no effect on the implanted organ, but would act immediately toreject the newly introduced bone marrow. Anti-human ATG obtained fromany mammalian host can be used, e.g., ATG produced in pigs, althoughthus far preparations of pig ATG have been of lower titer thanhorse-derived ATG. ATG is superior to anti-NK monoclonal antibodies, asthe latter are generally not lytic to all host NK cells, while thepolyclonal mixture in ATG is capable of lysing all host NK cells.Anti-NK monoclonal antibodies can, however, be used.

The presence of donor antigen in the host thymus during the time whenhost T cells are regenerating post-transplant is critical for tolerizinghost T cells. If donor hematopoietic stem cells are not able to becomeestablished in the host thymus and induce tolerance before host T cellsregenerate repeated doses of anti-recipient T cell antibodies may benecessary throughout the non-myeloablative regimen. Continuous depletionof host T cells may be required for several weeks.

It may also be necessary or desirable to splenectomize the recipient inorder to avoid anemia.

Second, natural antibodies are absorbed from the recipient's blood byhemoperfusion. Pre-formed natural antibodies (nAB) are the primaryagents of graft rejection. Natural antibodies bind to xenogeneicendothelial cells. These antibodies are independent of any knownprevious exposure to antigens of the xenogeneic donor. The mechanism bywhich newly developing B cells are tolerized is unknown. An α1-3galactose linkage epitope-affinity matrix, e.g., in the form of anaffinity column, e.g., matrix bound linear B type VI carbohydrate, isuseful for removing anti-swine antibodies from the recipient's blood.

The third step in the non-myeloablative procedure is to supply donorspecific growth factors or cytokines, to promote engraftment of donorstem cells.

As liver is the major site of hematopoiesis in the fetus, fetal livercan also serve as an alternative to bone marrow as a source ofhematopoietic stem cells. The thymus is the major site of T cellmaturation. Each organ includes an organ specific stromal matrix thatcan support differentiation of the respective undifferentiated stemcells implanted into the host. Although adult thymus may be used, fetaltissue obtained sufficiently early in gestation is preferred because itis free from mature T lymphocytes which can cause GVHD. Fetal tissuesalso tend to survive better than adult tissues when transplanted. As anadded precaution against GVHD, thymic stromal tissue can be irradiatedprior to transplantation, e.g., irradiated at 1000 rads. As analternative or an adjunct to implantation, fetal liver cells can beadministered in fluid suspension.

Fourth, bone marrow cells (BMC), or another source of hematopoietic stemcells, e.g., a fetal liver suspension, of the donor are injected intothe recipient. Donor BMC home to appropriate sites of the recipient andgrow contiguously with remaining host cells and proliferate, forming achimeric lymphohematopoietic population. By this process, newly formingB cells (and the antibodies they produce) are exposed to donor antigens,so that the transplant will be recognized as self. Tolerance to thedonor is also observed at the T cell level in animals in whichhematopoietic stem cell, e.g., BMC, engraftment has been achieved. Whenan organ graft is placed in such a recipient several months after bonemarrow chimerism has been induced, natural antibody against the donorwill have disappeared, and the graft should be accepted by both thehumoral and the cellular arms of the immune system. This approach hasthe added advantage of permitting organ transplantation to be performedsufficiently long following transplant of hematopoietic cells, e.g.,BMT, e.g., a fetal liver suspension, that normal health andimmunocompetence will have been restored at the time of organtransplantation. The use of xenogeneic donors allows the possibility ofusing bone marrow cells and organs from the same animal, or fromgenetically matched animals.

Many of the methods discussed in the art use whole body irradiation, tocreate hematopoietic space and thereby promote engraftment. The need forirradiation can be substantially reduced or eliminated by administeringa sufficient number of donor bone marrow cells. This should be combinedwith a treatment, e.g., thymic irradiation, which induces thymic space.

Finally, T cells, particularly, thymic or lymph node T cells, can befurther suppressed by administering to the recipient a short course ofan immunosuppressive agent, e.g., cyclosporine.

While any of these procedures may aid the survival of an implantedorgan, best results are achieved when all steps are used in combination.Methods of the invention can be used to confer tolerance to allogeneicgrafts, e.g., wherein both the graft donor and the recipient are humans,and to xenogeneic grafts, e.g., wherein the graft donor is a nonhumananimal, e.g., a swine, e.g., a miniature swine, and the graft recipientis a primate, e.g., a human.

In the case of xenogeneic grafts, the donor of the implant and theindividual that supplies either the tolerance-inducing hematopoieticcells or the liver to be perfused should be the same individual orshould be as closely related as possible. For example, it is preferableto derive implant tissue from a colony of donors that is highly inbred.

Detailed Protocol

In the following protocol for preparing a cynomolgus monkey for receiptof a kidney from a miniature swine donor, zero time is defined as themoment that the arterial and venous cannulas of the recipient areconnected to the liver to be perfused.

On day −1 a commercial preparation (Upjohn) of horse anti-humananti-thymocyte globulin (ATG) is injected into the recipient. ATGeliminates mature T cells and natural killer cells that would otherwisecause rejection of the bone marrow cells used to induce tolerance. Therecipient is anesthetized, an IV catheter is inserted into therecipient, and 6 ml of heparinized whole blood are removed beforeinfusion. The ATG preparation is then injected (50 mg/kg) intravenously.Six ml samples of heparinized whole blood are drawn for testing at timepoints of 30 min., 24 hours and 48 hours. Blood samples are analyzed forthe effect of antibody treatment on natural killer cell activity(testing on K562 targets) and by FACS analysis for lymphocytesubpopulations, including CD4, CD8, CD3, CD11b, and CD16. Preliminarydata from both assays indicate that both groups of cells are eliminatedby the administration of ATG. If mature T cells and NK cells are noteliminated, ATG can be re-administered at later times in the procedure,both before and after organ transplantation.

Sublethal irradiation administered in many art methods is omitted byincreasing the number of stem cells administered and by administering700 rads of thymic irradiation. Thymic irradiation is delivered on day0.

Natural antibodies are a primary cause of organ rejection. To removenatural antibodies from the recipient's circulation prior totransplantation, on day 0 an operative absorption of natural antibodies(nAB) is performed, using a miniature swine liver, as follows. At −90minutes the swine donor is anesthetized, And the liver prepared forremoval by standard operative procedures. At −60 minutes the recipientmonkey is anesthetized. A peripheral IV catheter is inserted, and a 6 mlsample of whole blood is drawn. Through mid-line incision, the abdominalaorta and the vena cava are isolated. Silastic cannulas containing sideports for blood sampling are inserted into the blood vessels.

At −30 minutes the liver is perfused in situ until it turns pale, andthen removed from the swine donor and placed into cold Ringers Lactate.The liver is kept cold until just prior to reperfusion in the monkey. Aliver biopsy is taken. At −10 minutes the liver is perfused with warmalbumin solution until the liver is warm (37 degrees).

At 0 time the arterial and venous cannulas of the recipient areconnected to the portal vein and vena cava of the donor liver andperfusion is begun. Liver biopsies are taken at 30 minutes and 60minutes, respectively. Samples of recipient blood are also drawn forserum at 30 minutes and 60 minutes respectively. At 60 minutes the liveris disconnected from the cannulas and the recipient's large bloodvessels are repaired. The liver, having served its function of absorbingharmful natural antibodies from the recipient monkey, is discarded.Additional blood samples for serum are drawn from the recipient at 2,24, and 48 hours. When this procedure was performed on two sequentialperfusions of swine livers, the second liver showed no evidence of mildischemic changes during perfusion.

To promote long-term survival of the implanted organ through T-cell andB-cell mediated tolerance, donor bone marrow cells are administered tothe recipient to form chimeric bone marrow. The presence of donorantigens in the bone marrow allows newly developing B cells, and newlysensitized T cells, to recognize antigens of the donor as self, andthereby induces tolerance for the implanted organ from the donor. Tostabilize the donor BMC, donor stromal tissue, in the form of tissueslices of fetal liver, thymus, and/or fetal spleen are transplantedunder the kidney capsule of the recipient. Stromal tissue is preferablyimplanted simultaneously with, or prior to, administration ofhematopoietic stem cells, e.g., BMC, or a fetal liver cell suspension.Sufficient stem cells are administered to eliminate the need forpreparative or hematopoietic space-creating irradiation.

To follow chimerism, two color flow cytometry can be used. This assayuses monoclonal antibodies to distinguish between donor class I majorhistocompatibility antigens and leukocyte common antigens versusrecipient class I major histocompatibility antigens. BMC can in turn beinjected either simultaneously with, or preceding, organ transplant.Bone marrow is harvested and injected intravenously as previouslydescribed (Pennington et al., 1988, Transplantation 45:21-26). Shouldnatural antibodies be found to recur before tolerance is induced, andshould these antibodies cause damage to the graft, the protocol can bemodified to permit sufficient time following BMT for humoral toleranceto be established prior to organ grafting.

The approaches described above are designed to synergistically preventthe problem of transplant rejection.

The methods of the invention may be employed in combination, asdescribed, or in part

The method of introducing bone marrow cells may be altered, particularlyby (1) increasing the time interval between injecting hematopoietic stemcells and implanting the graft; (2) increasing the amount ofhematopoietic stem cells injected; (3) varying the number ofhematopoietic stem cell injections; (4) varying the method of deliveryof hematopoietic stem cells; (5) varying the tissue source ofhematopoietic stem cells, e.g., a fetal liver cell-suspension may beused; or (6) varying the donor source of hematopoietic stem cells.Although hematopoietic stem cells derived from the graft donor arepreferable, hematopoietic stem cells may be obtained from otherindividuals or species, or from genetically-engineered inbred donorstrains, or from in vitro cell culture.

Methods of preparing the recipient for transplant of hematopoietic stemcells may be varied. For instance, recipient may undergo a splenectomy.The latter would preferably be administered prior to thenon-myeloablative regimen, e.g., at day −14.

Hemoperfusion of natural antibodies may: (1) make use of other vascularorgans, e.g., liver, kidney, intestines; (2) make use of multiplesequential organs or affinity matrices; (3) vary the length of time eachorgan or affinity matrices is perfused; (4) vary the donor of theperfused organ. Antibodies introduced prior to hematopoietic celltransplant may be varied by: (1) using monoclonal antibodies to T cellsubsets or NK cells (e.g., anti-NKH1_(A), as described by U.S. Pat. No.4,772,552 to Hercend, et al., hereby incorporated by reference); (2)preparing anti-human ATG in other mammalian hosts (e.g., monkey, pig,rabbit, dog); or (3) using anti-monkey ATG prepared in any of the abovementioned hosts.

The methods of the invention may be employed with other mammalianrecipients (e.g., rhesus monkeys) and may use other mammalian donors(e.g., primates, sheep, or dogs).

As an alternative or adjunct to hemoperfusion, host antibodies can bedepleted by administration of an excess of hematopoietic cells.

Stromal tissue introduced prior to hematopoietic cell transplant, e.g.,BMT, may be varied by: (1) administering the fetal liver and thymustissue as a fluid cell suspension; (2) administering fetal liver orthymus stromal tissue but not both; (3) placing a stromal implant intoother encapsulated, well-vascularized sites, or (4) using adult thymusor fetal spleen as a source of stromal tissue.

OTHER EMBODIMENTS

The methods described herein for inducing tolerance to, or promoting theacceptance of, an allogeneic antigen or allogeneic graft can be usedwhere, as between the donor and recipient, there is any degree ofmismatch at MHC loci or other loci which influence graft rejection.Preferably, there is a mismatch at at least one MHC locus or at at leastone other locus that mediates recognition and rejection, e.g., a minorantigen locus. With respect to class I and class II MHC loci, the donorand recipient can be: matched at class I and mismatched at class II;mismatched at class I and matched at class II; mismatched at class I andmismatched at class II; matched at class I, matched at class II. In anyof these combinations other loci which control recognition andrejection, e.g., minor antigen loci, can be matched or mismatched. Asstated above, it is preferable that there is mismatch at least onelocus. Mismatched at MHC class I means mismatched for one or more MHCclass I loci, e.g., in the case of humans, mismatched at one or more ofHLA-A, HLA-B, or HLA-C, or in the case of swine, mismatch at one or moreSLA class I loci, e.g., the swine A or B loci. Mismatched at MHC classII means mismatched at one or more MHC class II loci, e.g., in the caseof humans, mismatched at one or more of a DP α, a DPβ, a DQ α, a DQ β, aDR α, or a DR β, or in the case of swine, mismatch at one or SLA classII loci, e.g., mismatch at DQ α or β, or DR α or β.

The methods described herein for inducing tolerance to an allogeneicantigen or allogeneic graft can be used where, as between the donor andrecipient, there is any degree of reactivity in a mixed lymphocyteassay, e.g., wherein there is no, low, intermediate, or high mixedlymphocyte reactivity between the donor and the recipient. In preferredembodiments mixed lymphocyte reactivity is used to define mismatch forclass II, and the invention includes methods for performing allogeneicgrafts between individuals with any degree of mismatch at class II asdefined by a mixed lymphocyte assay. Serological tests can be used todetermine mismatch at class I or II loci and the invention includesmethods for performing allogeneic grafts between individuals with anydegree of mismatch at class I and or II as measured with serologicalmethods. In a preferred embodiment, the invention features methods forperforming allogeneic grafts between individuals which, as determined byserological and or mixed lymphocyte reactivity assay, are mismatched atboth class I and class II.

The methods of the invention are particularly useful for replacing atissue or organ afflicted with a neoplastic disorder, particularly adisorder which is resistant to normal modes of therapy, e.g.,chemotherapy or radiation therapy. Methods of the invention can be usedfor inducing tolerance to a graft, e.g., an allograft, e.g., anallograft from a donor which is mismatched at one or more class I loci,at one or more class II loci, or at one or more loci at each of class Iand class II. In preferred embodiments: the graft includes tissue fromthe digestive tract or gut, e.g., tissue from the stomach, or boweltissue, e.g., small intestine, large intestine, or colon; the graftreplaces a portion of the recipient's digestive system e.g., all or partof any of the digestive tract or gut, e.g., the stomach, bowel, e.g.,small intestine, large intestine, or colon.

Methods of the invention minimize or eliminate the need for preparativeWB irradiation. However, when irradiation is administered, it ispossible to induce mixed chimerism with less radiation toxicity byfractionating the radiation dose, i.e., by delivering the radiation intwo or more exposures or sessions. Accordingly, in any method of theinvention calling for the irradiation of a recipient, e.g., a primate,e.g., a human, recipient, of a xenograft or allograft, the radiation caneither be delivered in a single exposure, or more preferably, can befractionated into two or more exposures or sessions. The sum of thefractionated dosages is preferably equal, e.g., in rads or Gy, to theradiation dosage which can result in mixed chimerism when given in asingle exposure. The fractions are preferably approximately equal indosage. Hyperfractionation of the radiation dose can also be used inmethods of the invention. The fractions can be delivered on the sameday, or can be separated by intervals of one, two, three, four, five, ormore days. Whole body irradiation, thymic irradiation, or both, can befractionated.

Thymic irradiation can also be fractionated. For example, a single doseof 700 rads can be replaced with, e.g., two fractions of 350 rads, orseven fractions of 100 rads.

Methods of the invention can include recipient splenectomy.

As is discussed herein, hemoperfusion, e.g., hemoperfusion with a donororgan, can be used to deplete the host of natural antibodies. Othermethods for depleting or otherwise inactivating natural antibodies canbe used with any of the methods described herein. For example, drugswhich deplete or inactivate natural antibodies, e.g., deoxyspergualin(DSG) (Bristol), or anti-IgM antibodies, can be administered to therecipient of an allograft or a xenograft. One or more of, DSG (orsimilar drugs), anti-IgM antibodies, and hemoperfilsion, can be used todeplete or otherwise inactivate recipient natural antibodies in methodsof the invention. DSG at a concentration of 6 mg/kg/day, i.v., has beenfound useful in suppressing natural antibody function in pig tocynomolgus kidney transplants.

In any of the methods described herein, particularly primate or clinicalmethods, it is preferable to form mixed chimerism as opposed to entirelyreplacing the recipient's stem cells with donor cells.

Alternative methods for the inactivation of thymic T cells are alsoincluded in embodiments of the invention. Some of the methods describedherein include the administration of thymic irradiation to inactivatehost thymic-T cells or to otherwise diminish the host's thymic-T cellmediated responses to donor antigens. It has been discovered that thethymic irradiation called for in allogeneic or xenogeneic methods of theinvention can be supplemented with, or replaced by, other treatmentswhich diminish (e.g., by depleting thymic-T cells and/or down modulatingone or more of the T cell receptor (TCR), CD4 co-receptor, or CD8co-receptor) the host's thymic-T cell mediated response. For example,thymic irradiation can be supplemented with, or replaced by, anti-T cellantibodies (e.g., anti-CD4 and/or anti-CD8 monoclonal antibodies)administered a sufficient number of times, in sufficient dosage, for asufficient period of time, to diminish the host's thymic-T cell mediatedresponse.

For best results, anti-T cell antibodies should be administeredrepeatedly. E g., anti-T cell antibodies can be administered one, two,three, or more times prior to donor bone marrow transplantation.Typically, a pre-bone marrow transplantation dose of antibodies will begiven to the patient about 5 days prior to bone marrow transplantation.Additional, earlier doses 6, 7, or 8 days prior to bone marrowtransplantation can also be given. It may be desirable to administer afirst treatment then to repeat pre-bone marrow administrations every 1-5days until the patient shows excess antibodies in the serum and about99% depletion of peripheral T cells and then to perform the bone marrowtransplantation. Anti-T cell antibodies can also be administered one,two, three, or more times after donor bone marrow transplantation.Typically, a post-bone marrow transplant treatment will be given about2-14 days after bone marrow transplantation. The post bone marrowadministration can be repeated as many times as needed. If more than oneadministration is given the administrations can be spaced about 1 weekapart. Additional doses can be given if the patient appears to undergoearly or unwanted T cell recovery. Preferably, anti-T cell antibodiesare administered at least once (and preferably two, three, or moretimes) prior to donor bone marrow transplantation and at least once (andpreferably two, three, or more times) after donor bone marrowtransplantation.

Some of the methods herein include the administration of hematopoieticstem cells to a recipient. In many of those methods, hematopoietic stemcells are administered prior to or at the time of the implantation of agraft (an allograft or a xenograft), the primary purpose of theadministration of hematopoietic stem cells being the induction oftolerance to the graft. The inventors have found that one or moresubsequent administrations (e.g., a second, third, fourth, fifth, orfurther subsequent administration) of hematopoietic stem cells can bedesirable in the creation and/or maintenance of tolerance. Thus, theinvention also includes methods in which hematopoietic stem cells areadministered to a recipient, e.g., a primate, e.g., a human, which haspreviously been administered hematopoietic stem cells as part of any ofthe methods referred to herein.

While not wishing to be bound by theory the inventor believes thatrepeated stem cell administration may promote chimerism and possiblylong-term deletional tolerance in graft recipients. Accordingly, anymethod referred to herein which includes the administration ofhematopoietic stem cells can further include multiple administrations ofstem cells. In preferred embodiments: a first and a secondadministration of stem cells are provided prior to the implantation of agraft; a first administration of stem cells is provided prior to theimplantation of a graft and a second administration of stem cells isprovided at the time of implantation of the graft. In other preferredembodiments: a first administration of stem cells is provided prior toor at the time of implantation of a graft and a second administration ofstem cells is provided subsequent to the implantation of a graft. Theperiod between administrations of hematopoietic stem cells can bevaried. In preferred embodiments a subsequent administration ofhematopoietic stem cell is provided: at least two days, one week, onemonth, or six months after the previous administration of stem cells; atleast two days, one week, one month, or six months after theimplantation of the graft.

The method can further include the step of administering a second orsubsequent dose of hematopoietic stem cells: when the recipient beginsto show signs of rejection, e.g., as evidenced by a decline in functionof the grafted organ, by a change in the host donor specific antibodyresponse, or by a change in the host lymphocyte response to donorantigen; when the level of chimerism decreases; when the level ofchimerism falls below a predetermined value; when the level of chimerismreaches or falls below a level where staining with a monoclonal antibodyspecific for a donor PBMC antigen is equal to or falls below stainingwith an isotype control which does not bind to PBMC's, e.g. when thedonor specific monoclonal stains less than 1-2% of the cells; orgenerally, as is needed to maintain tolerance or otherwise prolong theacceptance of a graft. Thus, method of the invention can be modified toinclude a further step of determining if a subject which has received aone or more administrations of hematopoietic stem cells is in need of asubsequent administration of hematopoietic stem cells, and if so,administering a subsequent dose of hematopoietic stem cells to therecipient.

Any of the methods referred to herein can include the administration ofagents, e.g., 15-deoxyspergualin, mycophenolate mofetil, brequinarsodium, or similar agents, which inhibit the production, levels, oractivity of antibodies in the recipient. One or more of these agents canbe administered: prior to the implantation of donor tissue, e.g., one,two, or three days, or one, two, or three weeks before implantation ofdonor tissue; at the time of implantation of donor tissue; or afterimplantation of donor tissue, e.g., one, two, or three days, or one, twoor three weeks after, implantation of a graft.

The administration of the agent can be initiated: when the recipientbegins to show signs of rejection, e.g., as evidenced by a decline infunction of the grafted organ, by a change in the host donor specificantibody response, or by a change in the host lymphocyte response todonor antigen; when the level of chimerism decreases; when the level ofchimerism falls below a predetermined value; when the level of chimerismreaches or falls below a level where staining with a monoclonal antibodyspecific for a donor PBMC antigen is equal to or falls below stainingwith an isotype control which does not bind to PBMC's, e.g. when thedonor specific monoclonal stains less than 1-2% of the cells; orgenerally, as is needed to maintain tolerance or otherwise prolong theacceptance of a graft.

The period over which the agent is administered (or the period overwhich clinically effective levels are maintained in the subject) can belong term, e.g., for six months or more or a year or more, or shortterm, e.g., for less than a year, more preferably six months or less,more preferably one month or less, and more preferably two weeks orless. The period will generally be at least about one week andpreferably at least about two weeks in duration. In preferredembodiments the period is two or three weeks long.

Preferred embodiments include administration of 15-deoxyspergualin (6mg/kg/day) for about two weeks beginning on the day of graftimplantation.

Some of the methods referred to herein include the administration ofhematopoietic stem cells to a recipient. The inventors have found thatadministration of one or more cytokines, preferably a cytokine from thespecies from which the stem cells are derived, can promote engraftment,mixed chimerism, and tolerance, or otherwise prolong acceptance of agraft. The use of such cytokines can reduce or eliminate the need forwhole body irradiation. Thus, the invention also includes methods in therecipient is administered one or more cytokine, e.g., a donor-speciescytokine.

Although not wishing to be bound by theory, the inventors believe thatthe cytokines, particularly donor species cytokines, promote theengraftment and/or function of donor stem cells or their progeny cells.Accordingly, any method referred to herein which includes theadministration of hematopoietic stem cells can further include theadministration of a cytokine, e.g., SCF, IL-3, or GM-CSF. In preferredembodiments the cytokine one which is species specific in itsinteraction with target cells.

Administration of a cytokine can begin prior to, at, or after theimplantation of a graft or the implantation of stem cells.

The method can further include the step of administering a first orsubsequent dose of a cytokine to the recipient: when the recipientbegins to show signs of rejection, e.g., as evidenced by a decline infunction of the grafted organ, by a change in the host donor specificantibody response, or by a change in the host lymphocyte response todonor antigen; when the level of chimerism decreases; when the level ofchimerism falls below a predetermined value; when the level of chimerismreaches or falls below a level where staining with a monoclonal antibodyspecific for a donor PBMC antigen is equal to or falls below stainingwith an isotype control which does not bind to PBMC's, e.g. when thedonor specific monoclonal stains less than 1-2% of the cells; orgenerally, as is needed to maintain tolerance or otherwise prolong theacceptance of a graft. Thus, method of the invention can be modified toinclude a further step of determining if a subject is in need ofcytokine therapy and if so, administering a cytokine.

The period over which the cytokine(s) is administered (or the periodover which clinically effective levels are maintained in the subject)can be long term, e.g., for six months of more or a year or more, orshort term, e.g., for a year or less, more preferably six months orless, more preferably one month or less, and more preferably two weeksor less. The period will generally be at least about one week andpreferably at least about two weeks in duration.

In preferred embodiments the recipient is a primate, e.g., a human, andthe donor is from a different species, e.g., the donor is a pig and: pigSCF is administered; pig IL-3 is administered; a combination of pig SCFand pig IL-3 is administered; a pig specific hematopoiesis enhancingfactor, e.g., pig GM-SCF, is administered, e.g., after the implantationof stem cells, e.g., about a month after the implantation of stem cells.

A particularly preferred embodiment combines a short course, e.g., abouta month, of cyclosporine or a similar agent, a short course, e.g., abouttwo weeks, of 15-deoxyspergualin or a similar agent, and a short course,e.g., about two weeks, of donor specific cytokines, e.g., SCF and IL-3.In Cynomolgus monkeys receiving pig grafts and pig stem cells, treatmentwhich included the combination of cyclosporine (15 mg/kg/day for 28days), 15-deoxyspergualin (6 mg/kg/day for two weeks), and recombinantpig cytokines (SCF and IL-3, each at 10 μg/kg/day, i.v., for two weeks)was found to be useful. Administration began at the time of graftimplant. (The monkeys were also given a preparative regime consisting of3×100 cGy whole body irradiation on day −6, and −5 and hemoperfusionwith a pig liver just prior to stem cell administration.)

An anti-CD2 antibody, preferably a monoclonal, e.g., BTI-322, or amonoclonal directed at a similar or overlapping epitope, can be used inaddition to or in place of any anti-T cell antibodies (e.g., ATG) in anymethod referred to herein.

Other embodiments are within the following claims.

What is claimed is:
 1. A method of promoting tolerance in a recipientmammal of a first species, to a graft obtained from a donor mammal of asecond species comprising: (a) introducing hematopoietic stem cells ofsaid second species into said recipient mammal; (b) creating thymicspace in said recipient; (c) depleting or lysing donor-reactive T cellsof said recipient mammal; and (d) introducing said graft into saidrecipient, wherein the number of donor stem cells administered issufficient such that mixed chimerism can be formed without hematopoieticspace-creating irradiation.
 2. The method of claim 1 wherein said mixedchimerism is formed in the absence of hematopoietic space created bywhole body irradiation.
 3. The method of claim 1, wherein said recipientis a human.
 4. The method of claim 1, wherein said recipient is a humanand said graft is from a swine.
 5. The method of claim 4, wherein saidswine is a miniature swine.
 6. The method of claim 1, wherein saidthymic space is created by administering to the recipient at least onetreatment selected from the group consisting of thymic irradiation,steroids, corticosteroids, brequinar, and an immune suppressant chemicalor drug.
 7. The method of claim 1, wherein said thymic space is createdby administering thymic irradiation to said recipient.
 8. The method ofclaim 1, wherein said thymic space is created by administering an immunesuppressant chemical or drug to said recipient.
 9. The method of claim1, wherein said thymic space is created by administering a short courseof cyclosporin to said recipient.
 10. The method of claim 1, whereinmultiple hematopoietic stem cell administrations are provided to saidrecipient.
 11. The method of claim 1, further comprising inactivatingdonor-reactive NK cells of said recipient mammal.
 12. The method ofclaim 1, wherein said graft comprises a kidney.
 13. The method of claim1, wherein said graft comprises a liver.